Clustering and genetic differentiation of the normocyte binding protein (nbpxa) of Plasmodium knowlesi clinical isolates from Peninsular Malaysia and Malaysia Borneo

Abstract

BackgroundThe zoonotic malaria parasite Plasmodium knowlesi has become an emerging threat to South East Asian countries particular in Malaysia. A recent study from Sarawak (Malaysian Borneo) discovered two distinct normocyte binding protein xa (Pknbpxa) types of P. knowlesi. In the present study, the Pknbpxa of clinical isolates from Peninsular Malaysia and Sabah (Malaysian Borneo) were investigated for the presence of Pknbpxa types and natural selection force acting on the gene.MethodBlood samples were collected from 47 clinical samples from Peninsular Malaysia (n = 35) and Sabah (Malaysian Borneo, n = 12) were used in the study. The Pknbpxa gene was successfully amplified and directly sequenced from 38 of the samples (n = 31, Peninsular Malaysia and n = 7, Sabah, Malaysian Borneo). The Pknbpxa sequences of P. knowlesi isolates from Sarawak (Malaysian Borneo) were retrieved from GenBank and included in the analysis. Polymorphism, genetic diversity and natural selection of Pknbpxa sequences were analysed using DNAsp v 5.10, MEGA5. Phylogentics of Pknbpxa sequences was analysed using MrBayes v3.2 and Splits Tree v4.13.1. The pairwise FST indices were used to determine the genetic differentiation between the Pknbpxa types and was calculated using Arlequin 3.5.1.3.ResultsAnalyses of the sequences revealed Pknbpxa dimorphism throughout Malaysia indicating co-existence of the two types (Type-1 and Type-2) of Pknbpxa. More importantly, a third type (Type 3) closely related to Type 2 Pknbpxa was also detected. This third type was found only in the isolates originating from Peninsular Malaysia. Negative natural selection was observed, suggesting functional constrains within the Pknbpxa types.ConclusionsThis study revealed the existence of three Pknbpxa types in Malaysia. Types 1 and 2 were found not only in Malaysian Borneo (Sarawak and Sabah) but also in Peninsular Malaysia. A third type which was specific only to samples originating from Peninsular Malaysia was discovered. Further genetic studies with a larger sample size will be necessary to determine whether natural selection is driving this genetic differentiation and geographical separation.

Plasmodium knowlesi clinical isolates from Malaysia show extensive diversity and strong differential selection pressure at the merozoite surface protein 7D (MSP7D)

“…This is an interesting finding because the pkmsp7D sequences used in this study is from the same genomic data [24]. Bifurcation of trees, indicating dimorphism and strong negative/purifying selection on invasion genes like DBPαII (PkDBPαII) [31], PkNBPXa [26], PkAMA1 [30] have been reported from clinical samples. This probably indicates that Pkmsp7D is not influenced by geographical origin of the parasite and both the parasite sub-populations were under strong host immune pressure at the central region similar to the non-repeat region in Pkcsp from Malaysia [51].…”

Three Divergent Subpopulations of the Malaria ParasitePlasmodium knowlesi

“…Furthermore, it is divergent from clusters 1 and 2, which account for all infections in Malaysian Borneo and apparently a minority of wild macaque infections in peninsular Malaysia. With smaller numbers of samples, recent studies on sequence diversity in genes encoding the normocyte binding protein ( Pknbpxa ) ( 10 ) and the Duffy binding protein ( PkDBP ) ( 30 ), as well as the 18S rRNA gene and the mitochondrial Cox1 gene, have suggested that parasites in peninsular Malaysia had probably diverged from those in Malaysian Borneo.…”

“…Human infections in Malaysian Borneo, the portion of Malaysia on the island of Borneo, have divergent genetic subpopulations that are seen in the different macaque species locally, indicating that 2 independent zoonoses may be occurring sympatrically ( 9 ). Noticeable geographic differentiation of parasites between Malaysian Borneo and peninsular Malaysia was also evident in microsatellite analysis; separate studies have revealed divergence between the 2 regions at unlinked genes encoding the normocyte binding protein ( 10 – 12 ) and the Duffy binding protein ( 13 , 14 ), as well as the 18S rRNA and mitochondrial cytochrome oxidase subunit 1 ( 15 ). Whole-genome sequencing has confirmed the presence of 2 divergent subpopulations of P. knowlesi in Malaysian Borneo and revealed a third divergent cluster of laboratory isolates maintained in laboratories since the 1960s; most of these were recorded to have originated from peninsular Malaysia ( 16 ).…”

Analysis of nuclear and organellar genomes of Plasmodium knowlesi in humans reveals ancient population structure and recent recombination among host-specific subpopulations

“…It also provides evidence that the two distinct genome dimorphs reflect adaptation to either of the two host macaque species, although no evidence of a complete barrier in primate host susceptibility was found [ 10 ]. A third genome cluster has been described from geographically distinct Peninsular Malaysia [ 11 , 12 , 13 , 14 ].…”